Erythropoietin, the cytokine required for erythrocyte production, contributes to muscle progenitor cell proliferation and survival in culture and animal models. Erythropoietin induces transcription factors GATA-4 and TAL1 expression in myoblasts and knock down of GATA-4 or TAL1 changes myogenic regulatory factor expression program and promotes the myogenic differentiation. Sirtuin 1 activity, a NAD+ dependent deacetylation enzyme, myoblast cellular NAD+ level and NAD+/NADH ratio are increased by erythropoietin stimulation of C2C12 myoblasts. Sirtuin 1 knock down reduced GATA-4 and TAL1 expression and impaired the erythropoietin response suggesting that erythropoietin activity in myoblasts is mediated through GATA-4, TAL1 and Sirtuin 1. GATA-4 targets Sirtuin 1 to the myogenin promoter while TAL1 represses MyoD binding to and activation of the myogenin promoter. These data suggest a new pathway of erythropoietin activity in skeletal myoblasts affecting myogenic differentiation through cross-talk among Sirtuin1, GATA-4 and TAL1.